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目的构建survivin特异性短发夹RNA(shRNA)的表达载体,探讨其对人膀胱移行细胞癌T24细胞的生物学行为的影响。方法将构建survivin特异shRNA表达载体转染T24细胞,RT-PCR、Western blot检测survivin mRNA及其蛋白的表达水平;通过细胞生长曲线、随机运动实验、Matrigel穿膜实验检测肿瘤细胞生长、运动及体外侵袭能力。结果经EcoRⅠ和HindⅢ双酶切和测序鉴定,成功筛选含目的DNA片段的重组载体pshRNA-survivin2,并成功转染T24细胞;转染pshRNA-survivin2后可显著抑制细胞中survivin mRNA及其蛋白的表达,抑制率分别为61.73%和73.27%;pshRNA-sur-vivin2组的细胞侵袭力与运动能力均有明显的下降,第3、5天时肿瘤细胞生长抑制率分别为59.13%、83.86%。结论应用pTZU6+1质粒载体构建survivin的shRNA表达载体,能有效抑制其survivin mRNA及其蛋白的表达,并可抑制T24细胞的增殖、运动及体外侵袭能力。
Objective To construct the survivin specific short hairpin RNA (shRNA) expression vector and investigate its effect on the biological behavior of human bladder transitional cell carcinoma T24 cells. Methods The survivin-specific shRNA expression vector was transfected into T24 cells. The expression of survivin mRNA and protein was detected by RT-PCR and Western blot. The cell growth curve, stochastic movement assay and Matrigel transmembrane membrane assay were used to detect the growth, Invasive ability. Results The recombinant plasmid pshRNA-survivin2 was successfully screened by restriction endonuclease digestion and sequencing of EcoRⅠand HindⅢ. The recombinant plasmid pshRNA-survivin2 was successfully transfected into T24 cells. Transfection of pshRNA-survivin2 significantly inhibited the expression of survivin mRNA and protein , And the inhibitory rates were 61.73% and 73.27% respectively. The cell invasiveness and motor ability of pshRNA-sur-vivin2 group were significantly decreased. The growth inhibition rates of tumor cells were 59.13% and 83.86% on day 3 and day 5, respectively. Conclusion The shRNA expression vector pTZU6 + 1 was constructed by using plasmid pTZU6 + 1, which can effectively inhibit the expression of survivin mRNA and protein and inhibit the proliferation, exercise and invasion ability of T24 cells in vitro.